Clostridium difficile is an anaerobic, spore-forming bacterium that represents the leading cause of hospital-acquired diarrhea and life-threatening pseudomembranous colitis. C. difficile-associate disease (CDAD) affects more than 750,000 individuals each year in the U.S. and is responsible for more deaths than all other intestinal infections combined. Disease is mediated by two toxins, A and B that are potently cytotoxic and disrupt colonic epithelial cells, leading to inflammation and hemorrhage. CDAD typically is precipitated by disruption of the normal colonic bacterial flora with antibiotics, which provide opportunities for C. difficile to become established. The dramatic rise in CDAD over the past decade has paralleled the increased use of broad-spectrum antibiotics. Recent outbreaks of hypervirulent strains have been reported around the globe. New treatment strategies are urgently needed to combat the worldwide epidemic of C. difficile infections. Our approach to addressing this need has been to discover novel antitoxin monoclonal antibodies (mAbs) that demonstrate unprecedented toxin-neutralizing activity and define unique neutralization epitopes on the C. difficile toxins. These mAbs are 100-fold more potent that current-generation mAbs against hypervirulent strains and are the first to demonstrate complete and durable protection in a stringent animal model of CDAD. Whereas current-generation mAbs have demonstrated clinical promise in preventing recurrent disease but not in treating active disease, our ultrapotent mAbs may have utility in managing a spectrum of CDAD manifestations, including cases of active, severe or life-threatening disease. Our overall goal in this project is to ready our novel mAbs for human testing while elucidating fundamental structure-function relationships within the C. difficile toxins. Our project thus directly addresses Thematic Area 2: "Translating Basic Science Discoveries into New and Better Treatments." Aim 1 of our project seeks to define the novel neutralization epitopes to the level of individual amino acids. This information will provide new insights into C. difficile toxicity and its inhibition. Aim 2 will examine our mAbs in a series of innovative preclinical efficacy studies that will significantly expand our understanding of the potential role of antitoxin mAbs in the management of CDAD. Specific Aims 3 and 4 describe translational activities that are essential steps in readying our novel mAbs for human testing, which will be conducted under separate funding. Success in this project is defined as (1) favorable outcomes in the proposed preclinical mechanistic, efficacy and safety studies;and (2) clearance by FDA for us to conduct first-in-humans studies of our novel mAbs. Success would represent a major milestone in the development of an innovative, antitoxin therapy with unique potential to address some of the most pressing challenges of the C. difficile epidemic. PUBLIC HEALTH RELEVANCE: Clostridium difficile is a spore-forming bacterium that is the leading cause of hospital-acquired diarrhea and is responsible for more deaths per year in the U.S. than all other intestinal infections combined. The dramatic rise in the incidence and severity of C. difficile infections over the past decade have paralleled the increased use of broad- spectrum antibiotics, which provide opportunities for C. difficile to become established. Recently, outbreaks of hypervirulent strains have been reported across the globe. Our approach to meeting this worldwide public health challenge has been to develop novel antitoxin antibodies with unprecedented toxin-neutralizing activity in the best available preclinical models. In this project, we seek to complete key studies to ready our antitoxin antibodies for clinical testing while providing fundamental insight into C. difficile toxicity and its inhibition.